Processivity of cellulases and chitinases
Date
2017-02-14
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Abstract
Kahanevate naftavarude ja kasvava inimeste populatsiooni tõttu otsitakse võimalusi kasutada alternatiivseid energiaallikaid. Üheks võimaluseks on kasutada taastuva biokütuse ja tarbekaupade toorainena tselluloosi ja kitiini, mis on enim levinud biopolümeerid Maal. Lagundades tselluloos ja kitiin monomeerideks (vastavalt glükoosiks ja N-atsetüülglükoosamiiniks), on võimalik need edasi konverteerida bioetanooliks või kasutada neid oluliste kemikaalide sünteesiks.
Looduses lagundatakse tselluloos ja kitiin ensüümide abil. Vastavalt viivad tselluloosi lagundamist läbi ensüümid, mida nimetakse tsellulaasideks ja kitiin degradeeritakse kitinaaside abil. Ensümaatilist hüdrolüüsi rakendatakse ka tööstuses, et vältida kontsentreeritud mineraalhapete ja kõrgemate temperatuuride kasutamist. Kahjuks on looduslikud ensüümid madala efektiivsusega, mistõttu ensüümide tootmisega seotud kulutused moodustavad suure osa kujunevast toodangu lõpphinnast. Ensüümide võimekust on aga võimalik parandada insenergeneetilise modifitseerimise teel. Kuid vaatamata sellele, et tsellulaase ja kitinaase uuritakse üle poole sajandi, on teadmised ensüümide toimimise molekulaarsetest mehhanismidest veel poolikud.
Üheks oluliseks tsellulaaside ja kitinaaside kineetiliseks parameetriks on protsesiivsus, mis väljendub keskmise katalüüsi aktide arvuna, mille ensüüm sooritab enne substraadilt lahkumist. Tasub ka mainida, et peamiselt panustavad just protsessiivsed ensüümid tselluloosi/kitiini lagundamisele, mistõttu on nende toimemehhanismide väljaselgitamine kriitilise tähtsusega.
Käesoleva töö eesmärk oli protsessivsete tsellulaaside ja kitinaaside biokeemiline kirjeldamine. Töö raames töötati välja uued meetodid protsessiivsuse ja teiste kineetiliste parameetrite mõõtmiseks. Eelnevalt oli teada, et individuaalse protsessiivse tsellulaasi hüdrolüüsi kiirust limiteerivaks faktoriks on ensüümi aeglane dissotsieerumine substraadilt. Antud töös aga selgus, et koostöös teiste tsellulaasidega on hüdrolüüsi kiirus piiratud ensüümi protsessiivse liikumise kiiruse poolt.
Lisaks leidsime, et efektiivseks hüdrolüüsi toimumiseks on olulised tugevad ensüüm-substraat interaktsioonid ensüümi aktiivtsentri substraadi sidumiskohtades ja tugevad ensüüm-produkt interaktsioonid aktiivtsentri produkti sidumiskohtades. Ühe interaktsiooni kadumine viib hüdrolüüsi efektiivsuse järsule langusele.
Dwindling oil reserves and increasing world population necessitate the search for alternative sources of energy. Thus, cellulose and chitin, the net annual production of which are estimated in billions of tons, have recently become potential candidates as a renewable sources for the production of biofuel for gasoline working vehicles as well as essential chemical compounds for food and pharmaceutical industries. The main idea lies in degradation of cellulose and chitin into monomers (i.e. glucose and N- acetylglucosamine respectively). The latter can be easily converted into ethanol and other chemicals. In nature cellulose is degraded by enzymes with common name cellulases and chitin is degraded by enzymes called chitinases. Cellulases and chitinases are also employed for industrial uses since they enable to avoid the use of high temperatures and concentrated mineral acids. However, natural enzymes are slow and their contribution to the cost of the end products remains high. Efficient degradation of polysaccharides is achieved by concerted action of different types of enzymes. The main contribution to this process generally comes from processive enzymes that constitute the major part of cellulolytic/chitinolytic systems. Processivity is defined as the ability of an enzyme to conduct sequential catalytic acts without dissociating from its substrate. It is therefore an important characteristic of cellulose/chitin degrading enzymes, and its investigation is likely to provide a good clue to the improvement of the process of saccharification of recalcitrant polysaccharides. The present study was aimed at biochemical description of processive cellulases and chitinases. New methods for the measurement of processivity and other kinetic parameters were developed. It was previously know that the limiting step in the catalytic cycle of enzyme acting in isolation is the dissociation from the substrate. On the contrary we found that the limiting step in the catalytic cycle of enzyme acting in the synergistic mixture is the processive movement along the polymer chain. We also demonstrated that for the efficient degradation of recalcitrant polysaccharides strong interactions in the substrate binding site and in the product binding site are required. The loss of one interaction leads to reduced activity on crystalline substrates but concomitantly increases activity on amorphous substrates.
Dwindling oil reserves and increasing world population necessitate the search for alternative sources of energy. Thus, cellulose and chitin, the net annual production of which are estimated in billions of tons, have recently become potential candidates as a renewable sources for the production of biofuel for gasoline working vehicles as well as essential chemical compounds for food and pharmaceutical industries. The main idea lies in degradation of cellulose and chitin into monomers (i.e. glucose and N- acetylglucosamine respectively). The latter can be easily converted into ethanol and other chemicals. In nature cellulose is degraded by enzymes with common name cellulases and chitin is degraded by enzymes called chitinases. Cellulases and chitinases are also employed for industrial uses since they enable to avoid the use of high temperatures and concentrated mineral acids. However, natural enzymes are slow and their contribution to the cost of the end products remains high. Efficient degradation of polysaccharides is achieved by concerted action of different types of enzymes. The main contribution to this process generally comes from processive enzymes that constitute the major part of cellulolytic/chitinolytic systems. Processivity is defined as the ability of an enzyme to conduct sequential catalytic acts without dissociating from its substrate. It is therefore an important characteristic of cellulose/chitin degrading enzymes, and its investigation is likely to provide a good clue to the improvement of the process of saccharification of recalcitrant polysaccharides. The present study was aimed at biochemical description of processive cellulases and chitinases. New methods for the measurement of processivity and other kinetic parameters were developed. It was previously know that the limiting step in the catalytic cycle of enzyme acting in isolation is the dissociation from the substrate. On the contrary we found that the limiting step in the catalytic cycle of enzyme acting in the synergistic mixture is the processive movement along the polymer chain. We also demonstrated that for the efficient degradation of recalcitrant polysaccharides strong interactions in the substrate binding site and in the product binding site are required. The loss of one interaction leads to reduced activity on crystalline substrates but concomitantly increases activity on amorphous substrates.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
biokeemia, ensüümid, tsellulaasid, kitinaasid, biochemistry, enzymes, cellulases, chitinases